1. Field of the Invention
The present invention relates to a method for mutating genetic material and screening mutant genetic material for a desired mutation. The invention further relates to mutant genetic material produced according to the disclosed method, as well as to expression products of such mutant genetic material.
2. Description of the Background Art
The largest class of naturally occurring proteins is made up of enzymes. Each enzyme generally catalyzes a different kind of chemical reaction, and is usually highly specific in its function. An enzyme molecule contains an active site to which a specific substrate is bound during a catalytic cycle.
Although there may be slight variations in a distinct type of naturally occurring enzyme within a given species of organism, enzyme molecules of a specific type produced by organisms of the same species generally are substantially identical with respect to substrate specificity, thermal stability, activity levels under various conditions (e.g., temperature and pH), oxidation stability, and the like. Such characteristics of a naturally occurring or "wild-type" enzyme are not necessarily optimized for utilization outside of the natural environment of the enzyme. It may thus be desirable to alter a natural characteristic of an enzyme to optimize a certain property of the enzyme for a specific use, or for use in a specific environment
The amino acid sequence of an enzyme determines the characteristics of the enzyme, and the enzyme's amino acid sequence is specified by the nucleotide sequence of a gene coding for the enzyme. A change of the amino acid sequence of an enzyme may alter the enzyme's properties to varying degrees, or may even inactivate the enzyme, depending on the location, nature and/or magnitude of the change in the amino acid sequence.
Methods for introducing specific amino acid changes into proteins, such as oligonucleotide-directed mutagenesis of DNA, are known in the art. However, the ability to predict the effects of a specific mutation is poor, making the process of creating and characterizing desired mutations laborious.
Methods for random mutagenesis of genetic material are also known, but in the absence of a method for rapid and effective screening of a large number of mutants, identification of organisms having the desired mutation is tedious.
There remains a need in the art for new and practical mutagenesis and screening methods and for the products thereof.
Naturally occurring bacterial proteases are currently used for many purposes, among these is as an additive to washing preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains. Unfortunately, naturally-occurring proteases lose activity when stored in solution with detergents. Typically this decay of activity is geometric in nature, that is, a certain percentage of activity is lost in each time interval. The present invention provides a method to develop novel proteases with enhanced thermal stability and which survive prolonged storage in liquid detergents much longer than naturally-occurring proteases.